Molecular mechanisms of antigen retrieval: antigen retrieval reverses steric interference caused by formalin-induced cross-links.
Molecular mechanisms of antigen retrieval: antigen retrieval reverses steric interference caused by formalin-induced cross-links.
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DOI：
10.3109/10520290903039078
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发表时间：
2009-10
期刊：
影响因子：
--
通讯作者：
Sompuram SR
中科院分区：
文献类型：
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作者：
Bogen SA;Vani K;Sompuram SR
绝大多数用于福尔马林固定石蜡包埋（FFPE）组织的抗体需要抗原回收以逆转福尔马林固定的影响并重新建立免疫反应性。这种逆转是如何发生的，人们知之甚少。我们建立了一个新的实验模型来研究福尔马林固定和抗原回收的机制。对9种可用于FFPE组织的抗体的表位定位研究表明，每种抗体都由天然蛋白中的连续氨基酸组成（“线性表位”）。代表HER2、雌激素和孕激素受体抗体表位的小肽以肽阵列共价附着在玻璃显微镜载玻片上。大多数肽在福尔马林固定后仍保持免疫反应性。然而，如果在福尔马林诱导的交联过程中存在不相关的大蛋白，则所有肽的免疫反应性完全取消。我们假设不相关的蛋白与肽表位交联可以立体阻断抗体的结合。抗原回收解离不相关的蛋白质和恢复免疫反应性。由于临床抗体的表位只需要一级蛋白结构，因此抗原检索可能使蛋白质的二级和三级结构变性的事实是无关的。同样的机制可能发生在组织样品经受福尔马林固定和抗原回收。
The overwhelming majority of antibodies useful for formalin fixed, paraffin embedded (FFPE) tissues require antigen retrieval to reverse the effect of formalin fixation and re-establish immunoreactivity. How this reversal happens is poorly understood. We developed a new experimental model for studying the mechanism of formalin fixation and antigen retrieval. Epitope mapping studies on nine antibodies useful for FFPE tissues revealed that each consisted of a contiguous stretch of amino acids in the native protein (“linear epitope”). Small peptides representing the epitopes of antibodies to HER2, estrogen and progesterone receptors were attached covalently to glass microscope slides in a peptide array. Most peptides retained immunoreactivity after formalin fixation. Immunoreactivity was completely abrogated for all peptides, however, if an irrelevant large protein was present during formalin-induced cross-linking. We hypothesize that cross-linking the irrelevant protein to the peptide epitopes sterically blocked antibodies from bonding. Antigen retrieval dissociates irrelevant proteins and restores immunoreactivity. Because the epitopes for clinical antibodies require only primary protein structure, the fact that antigen retrieval probably denatures the secondary and tertiary structure of the protein is irrelevant. The same mechanism may occur in tissue samples subjected to formalin fixation and antigen retrieval.